Mutagenesis, B-gal, & 2-Hybrid Questions

S.L. Rennie rennie at bldghsc.lan1.umanitoba.ca
Fri Feb 21 14:16:46 EST 1997


In article <5ecuqp$2l at uni00nw.unity.ncsu.edu>, mike at ncsu.edu wrote:

> Hello,
> 
> I am conducting a 2-hybrid screen using strain PJ69.4a.  When I do a
> large scale transformation with decent efficiency I end up with a lot
> of cells.  I usually spin them down out of the PEG after the heat
> shock and resuspend in water, then plate them out.  The problem is
> that I am putting so many cells on each plate that they "glop up", so
> much so that it might impair the growth of positives or obscure their
> detection.  If I dilute the cells and use more plates then I will have
> to use *a lot* of plates, more than seems reasonable.  I am
> considering taking my transformation and letting it recover in drop
> out broth before plating, this won't cut down on the number of cells,
> but may enrich for positives.  I know a drawback here is that strong
> positives will be enriched for disproportionately, meaning that I
> would have to screen more of the positives to find out if there were
> multiple proteins interacting with the bait.  But right now anything
> at all would be welcome.  Any feed back on this situation would be
> welcome.  
> 
> Specifically, how do I get rid of all the dead/nontransformed cells
> before plating? 


Hi Mike
   Phil's strain has worked really well for our library screens.  The
convenience of this strain is that the reporter genes are stable
integrated and not on a plasmid at all  I've never  heard of people
complaining about too many transformants when trying to do a screen....How
many cells/plate do you think is too much?  We traditionally use 50 small
(yeast library) or 100 large (human library) to plate our screens and are
expecting 4 or 12 million transformants respectively
   And why would you need to get rid of dead/nontransformed cells, in our 
experience they don't inhibit the growth of true positives...we use SC -trp
-leu-his-ade 1mM 3-AT media to select for positives.


> 
> ***Also, I recently saw a seminar that had slides of blue yeast
> growing(?) on media.  Was the X-gal included in the media.  I thought
> that B-gal wasn't excreted by yeast and that they had to be lysed,
> what was going on here?

ON SC media with sucrose carbon source titred to pH 7 with KPO4 the Bgal
substrate is taken up by the yeast and the colonies turn
blue....apparently
the almighty demigods at Clontech don't like people to know this??  But it is
true that filter lifts are more sensitive.
> 
> 
> ***Another interesting thing that the guy was doing was PCR
> mutagenizing the components of a known positive interaction and then
> transforming the PCR product and the linearized vector into the yeast,
> where they spontaneously/naturally recombine.  Anybody else ever done
> this.  Seems like a great way to investigate specific interactions
> between your two proteins.

And yes homologous ends will recombine and form a closed plasmid in yeast at
a useful rate  this bit of knowledge has been out for over a decade, I don't 
know what the reference may be.


Sincerely
S. Rennie
astounded by the number of "yeast geneticists" relying on Clontech for info???  
>

-- 





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